Microbial single-cell RNA sequencing by split-pool barcoding

Abstract

Single-cell RNA-sequencing (scRNA-seq) has become an essential tool for characterizing multicelled eukaryotic systems but current methods are not compatible with bacteria. Here, we introduce microSPLiT, a low cost and high-throughput scRNA-seq method that works for gramnegative and gram-positive bacteria and can resolve transcriptional states that remain hidden at a population level. We applied microSPLiT to >25,000 Bacillus subtilis cells sampled from different growth stages, creating a detailed atlas of changes in metabolism and lifestyle. We not only retrieve detailed gene expression profiles associated with known but rare states such as competence and PBSX prophage induction, but also identify novel and unexpected gene expression states including heterogeneous activation of a niche metabolic pathway in a subpopulation of cells. microSPLiT empowers high-throughput analysis of gene expression in complex bacterial communities.

Efficient and cost-effective bacterial mRNA sequencing from low input samples through ribosomal RNA depletion

“…While we again observed rRNA depletion and a corresponding enrichment of mRNA compared to control samples, the effects were less pronounced with a less than 2-fold rRNA depletion, consistent with previous reports (Fig. S1) [14,15]. We hypothesize that this reduced efficiency arises from RNA degradation that might occur during the incubation at 37 o C for 1 hour or the additional cleanup step that is necessary prior to treatment with the poly-A polymerase.…”

“…As an alternate strategy, we also incorporated TEX treatment in EMBR-seq as it has previously been shown to specifically degrade rRNAs with 5'-monophosphate ends but not mRNAs that have 5'-triphosphate ends [10,14,15]. While we again observed rRNA depletion and a corresponding enrichment of mRNA compared to control samples, the effects were less pronounced with a less than 2-fold rRNA depletion, consistent with previous reports (Fig.…”

“…However, this method did not deplete rRNA, resulting in low mRNA detection efficiencies of ~0.5-2% (or ~40 mRNA per E. coli cell). In another study, Kuchina et al combined rRNA depletion with combinatorial barcoding to achieve ~5-10% mRNA detection efficiencies in B. subtilis [14]. These initial efforts suggest that improved…”

“…While this limitation of pre-designed kits have been overcome through the development of workflows to generate custom subtractive hybridization probe sets for any species of interest, they still operate at microgram quantities of starting material and either require multiple rounds of hybridization or a series of oligo optimization steps prior to optimal performance [12,13]. An alternate approach relies on the Terminator TM 5'-phosphate-dependent exonuclease (TEX) (Lucigen) to specifically degrade rRNAs with 5'-monophosphate ends but not mRNAs with 5'-triphosphate ends; however, this method typically has lower efficiencies than other existing rRNA depletion strategies [10,14,15]. A more recent method uses complementary single-stranded DNA probes to tile rRNAs that are subsequently degraded by RNase H [16].…”

Prokaryotic Single-Cell RNA Sequencing by In Situ Combinatorial Indexing

“…Most notably, a customized tagmentation protocol during library preparation could theoretically increase the mRNA capture by 2-fold (see "Future directions for optimization" in Methods). Following our initial deposit of an earlier version of this manuscript on biorXiv 42 , and during its formal review, Kuchina and colleagues deposited a manuscript on biorXiv 43 reporting a conceptually similar split-pool based bacterial scRNA-seq method in which in situ polyadenylation was utilized to capture mRNAs. It will be of great interest to compare and contrast these methods and to further improve the performance of PETRI-seq.…”